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  • Alkaline Phosphatase, Calf Intestinal (CIP)

    Description

    Alkaline Phosphatase, Calf Intestinal (CIP) nonspecifically catalyzes the dephosphorylation of 5´ and 3´ ends of DNA and RNA phosphomonoesters. Also, CIP hydrolyses ribo-, as well as deoxyribonucleoside triphosphates (NTPs and dNTPs). CIP is useful in many molecular biology applications such as the removal of phosphorylated ends of DNA and RNA for subsequent use in cloning or end-labeling of probes. In cloning, dephosphorylation prevents religation of linearized plasmid DNA. The enzyme acts on 5´ protruding, 5´ recessed and blunt ends. CIP may also be used to degrade unincorporated dNTPs in PCR reactions to prepare templates for DNA sequencing or SNP analysis.

    Product Source

    Calf intestinal mucosa

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    CutSmart™ Buffer-2010X

    Advantages and Features

    Applications

    • Dephosphorylation of cloning vector DNA to prevent recircularization during ligation
    • Dephosphorylation of DNA prior to end-labeling using T4 Polynucleotide Kinase
    • Treatment of dNTPs in PCR reactions prior to sequencing or SNP analysis
    • Dephosphorylation of DNA and RNA

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that hydrolyzes 1 μmol of p-Nitrophenyl Phosphate, PNPP (NEB #P0757) in a total reaction volume of 1 ml in 1 minute at 37°C.

    Reaction Conditions

    1X CutSmart™ Buffer
    Incubate at 37°C

    1X CutSmart™ Buffer:
    50 mM Potassium Acetate
    20 mM Tris-acetate
    10 mM Magnesium Acetate
    100 μg/ml BSA
    pH 7.9 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    50 mM KCl
    1 mM MgCl2
    50% Glycerol
    0.1 mM ZnCl2
    pH 8.2 @ 25°C

    Heat Inactivation

    No

    Molecular Weight

    Theoretical: 69 kDa

    Specific Activity

    3,000 units/mg

    Unit Assay Conditions

    1 M diethanolamine- HCl (pH 9.8), 0.5 mM MgCl2, 50 mM p-nitrophenylphosphate and enzyme. These conditions are only used for quantitating enzyme activity.

    Quality Control

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Functional Test (Dephosphorylation):
      The Alkaline Phosphatase is tested in a reaction with linearized DNA.  After incubation the DNA is ligated and transformed into competent cells.  The degree of dephosphorylation is determined by comparison to linearized DNA that is not treated with the alkaline phosphatase.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Notes

    1. CIP, as are most alkaline phosphatases, is a Zn2+ and Mg2+-dependent enzyme. Our formulation of its storage buffer provides Zn2+ and Mg2+, which does not require supplemental zinc or other additives in reactions with CIP.

    2. CIP is also active in 1X NEBuffers 1.1, 2.1, 3.1, as well as NEBuffers 1, 2, 3, 4 and unique NEBuffer for EcoRI.

    3. CIP activity is enhanced in the presence of monovalent salts.

    4. CIP is inhibited by metal chelators (e.g. EDTA), inorganic phosphate and phosphate analogs.

    References

    1. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, (2nd ed,). (p5.72), Cold Spring Harbor Laboratory Press .
    2. Mossner, E., Boll, M. and Pfleiderer, G. (1980). Hoppe Seylers Z. Physiol. Chem.. 361

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Which alkaline phosphatase, rSAP, CIP, or Antarctic Phosphatase, works best?
    2. The number of colonies that do not contain an insert seems high, how can I tell if the CIP worked?
    3. Will CIP work in restriction enzyme NEBuffers?
    4. Does the DNA need to be purified after the restriction digest, prior to CIP treatment?
    5. Does the DNA need to be purified after CIP treatment?
    6. Will CIP dephosphorylate proteins?
    7. Can Antarctic Phosphatase remove the 3' phosphate from DNA?
    8. Can CIP be heat inactivated?
    9. Why do some protocols recommend using CIP at 50°C and some recommend 37°C?
    1. Protocol for Dephosphorylation of 5´-ends of DNA using CIP in Restriction Enzyme Reaction (M0290)

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